Optical fluorescent probes of cellular structure and function are widely used in biology in fields as diverse as immunology, histochemistry, apoptosis, cell adhesion, chemotaxis, signal transduction, and electrophysiology. These probes include traditional chemically-based sensors and genetically-encoded fluorescent protein biosensors, all of which enable the measurement of physiological signals in real-time in living cells and tissue. For this purpose, cooled charge-coupled device (CCD) or photodiode arrays have typically been used with imaging optics. These commercial systems have limited sampling speed and limitations in the size of the imaging area, and are generally bulky and costly. A new method, Contact Fluorescence Imaging (CFI), is a simple solution for the spatiotemporal imaging of fluorescence signals over large areas. The challenge in terms of the widespread application of this method is the availability of a fast response, large format imager with high sensitivity. In the exploratory R21 phase, we will show feasibility of a new type of imager based on complementary metal oxide semiconductor (CMOS) technology. Advanced analog signal processing techniques will render the imager capable of pixel-level baseline subtraction, contrast mode operation, dual wavelength imaging, and ratiometric imaging, all capabilities that are lacking in commercially available imaging arrays. In the R33 phase, we will scale up the size of the sensor arrays, and produce a large format CMOS imager that can be tiled from smaller modular arrays. The outcome of this project will be a compact imaging system with the ability to monitor static and dynamic cellular events with fluorescent biosensors. It will be miniature, portable, rugged, reusable and adaptable to a wide range of applications. We will demonstrate how the new imaging system can be used to visualize the spatiotemporal dynamics of calcium and voltage waves in large-sized cultures of cardiac cells, as well as its general applicability as a conventional fluorescence imager that can be attached to the camera port of commercial microscopes.